CN1936019B - Probe encoding method for multiplex real-time nucleic acid amplification detection - Google Patents

Abstract

The invention relates to a probe coding method for multiple real time nucleic acid amplification testing that includes the following steps: using the fluorescent dye of nucleic acid amplification testing instrument as the basal element of fluorescent probe mark that is called fluorescent basal element; marking the specificity probe of target sequence between the fluorescent basal element inter-compounding. Using the quantity of fluorescent basal element, cycling threshold value and the relative intension of the basal elements, the random plural gene specificity fluorescent fingerprint atlas would be gained to testing plural different nucleic acid sequence synchronously under the situation of limited fluorescent basal element quantity. The invention is suited to test multi target sequence.

Description

Probe encoding method for multiplex real-time nucleic acid amplification detection

technical field

The present invention relates to a nucleic acid amplification detection, in particular to a probe coding method for multiple real-time nucleic acid amplification, which makes the number of target sequences detected by multiple real-time nucleic acid amplification much larger than the number of fluorescent dyes detectable by the instrument number. the

Background technique

Real-time polymerase chain reaction (Real-time PCR) is being more and more widely used in gene quantification, genotyping and detection of single nucleotide polymorphisms (SNPs). However, other detection technologies relying on nucleic acid amplification, regardless of the detection throughput and operation, are not as simple as real-time PCR, which is the root cause of the widespread clinical application of real-time PCR. However, real-time PCR has the problem of small detection capacity, that is, the number of target sequences that can be detected simultaneously in one reaction is limited by the number of fluorescence detection channels of the instrument. At present, the latest real-time PCR instrument can detect 5 different fluorescent dyes at the same time, and at most 5 target sequences can be detected according to the traditional detection method. This problem severely restricts the application of real-time PCR in multilocus-dependent genotyping. the

The simplicity and many other advantages of real-time PCR have attracted continuous efforts to increase its detection capacity. Since the Kramer group (Vet, J.A.M., et al, PNAS96, 6394-6399, 1999) took the lead in realizing the simultaneous detection of four target sequences on a four-color fluorescent PCR instrument, multi-component real-time PCR detection technology has been widely used. In 2000, Tyagi et al. (Tyagi, S., et al, Nature Biotechnology 18, 1191-1196, 2000) invented a wavelength-shifting molecular beacon, which overcomes the difficulty that a single light source cannot simultaneously detect multiple fluorescent dyes. El Hajj et al. A five-color probe was used to detect the rifampicin-resistant mutation of Mycobacterium tuberculosis (El Hajj, H.H., Journal of Clinical Microbiology39, 4131-4137, 2001). the

Leaving aside the real-time PCR instrument, Lee et al. (Lee, L.G., et al, Biotechniques27, 342-349, 1999) used a fluorescent photometer to scan 7 fluorescent dyes synchronously, realizing the simultaneous detection of 6 target sequences. In addition, the melting point difference of the PCR amplification product is used to analyze the melting point curve after the real-time PCR amplification is completed, so as to realize the simultaneous detection and genotyping of multiple target sequences (Herrmann, M.G., et al, Clinical Chemistry 50, 982, 2004; Elenitoba-Johnson, K.S.J., et al, Nature Medicine 7, 249-253, 2001; Ririe, K.M., et al, Analytical Biochemistry 245, 154-160, 1997). However, the limitation of this method is that the melting point range of PCR products is narrow, it is difficult to accommodate the simultaneous existence and distinction of more than 5 target sequences, and it is easily interfered by non-specific amplification such as primer dimers. the

Contents of the invention

The purpose of the present invention is aimed at the small detection capacity of multiple real-time nucleic acid amplification, that is, according to the traditional method of labeling a probe with a fluorescent group, the number of target sequences that can be detected simultaneously in one reaction is limited by the number of fluorescence detection channels of the instrument. The problem of limitation is to provide a probe encoding method or a probe combination method that enables multiplex real-time nucleic acid amplification to detect a significantly greater number of target sequences than the number of fluorescent dyes detectable by current instruments. the

Concrete steps of the present invention are:

1. Different fluorescent dyes that can be detected by nucleic acid amplification detection instruments are used as the basic elements of fluorescent probe labeling, which are called fluorescent basic elements;

2. Compounding or mixing specific indicator probes for labeling target sequences by the mutual combination of fluorescent basic elements. The mutual combination refers to combination code, permutation code and index code. the

The combination coding only considers the difference in the types of fluorescent basic elements and ignores the difference in fluorescence intensity between different fluorescent basic elements. The fluorescent basic elements can be combined into different fluorescent labeling groups or probes according to mathematical combination rules. The so-called arrangement coding considers the difference in the types of fluorescent basic elements and distinguishes the relative strength of each fluorescent basic element, and can be combined into different fluorescent labeling groups or probes according to mathematical arrangement rules. The said index coding takes into account the differences in the types of fluorescent basic elements, the relative strength of the fluorescence intensity among the elements, and the different degrees of the difference in fluorescence intensity, and can be combined into different fluorescent labeling groups or probes according to the mathematical index rules. . the

The specific indicator probes that use the combination of fluorescent basic elements instead of a single fluorescent basic element to compound or mix the target sequence; add the labeled probe to the real-time nucleic acid amplification system and test the nucleic acid The sequence is amplified; the three variables of the type of fluorescent basic elements appearing in the real-time nucleic acid amplification curve map, the cycle threshold ( _CT value) difference, and the relative intensity difference of each fluorescent basic element are used to distinguish and identify genes (signals) Specific fluorescent fingerprints and their corresponding target sequences for the simultaneous detection of multiple different nucleic acid sequences with a limited number of fluorescent basic elements. Said fluorescent basic elements include but not limited to ALEXA350, FAM, HEX, TET, JOE, VIC, ROX, Texas Red, Cy5, Cy5.5, TAMRA and so on.

Said fluorescent probes refer to fluorescent probes that can be used for real-time nucleic acid amplification detection, and they include TaqMan ^TM probes, TaqMan-MGB ^TM probes, molecular beacon probes (Molecular Beacons), fluorescence energy resonance transfer probes (also known as LightCycler ^TM probes), displacement probes, scorpion primers (Scorpions), Amplifier primers, etc. These fluorescent probes can be divided into probes that depend on the 5′→3′ exonucleic acid activity of the polymerase, such as TaqMan probes, and probes that do not depend on the 5′→3′ exonucleic acid activity of the polymerase, such as molecular beacons. probes and displacement probes, etc.

The nucleic acid amplification detection is a multicolor real-time nucleic acid amplification detection, or an end-point nucleic acid amplification detection.

The kind of said fluorescent basic elements refers to those fluorescent basic elements appearing in the real-time nucleic acid amplification curve atlas; said cycle threshold value ( _CT value) difference refers to the peak time between each fluorescent basic elements that appear The difference in fluorescence intensity between the fluorescent basic elements refers to the size level of each fluorescent basic element, or the fluorescence level, or the relative degree, or the relative intensity combined into different elements. The relative time of the peaks of various fluorescent basic elements, the shape of the curve, whether there is an inflection point of curve growth, etc. can be used to distinguish which sequence exists when multiple target sequences coexist and to amplify and detect.

The encoding method provided by the present invention is suitable for the detection of multiple target sequences, especially for genotyping that may only contain at least one (often one or a few) of multiple target sequences in the detection sample, especially is genotyping for different alleles in the same gene or gene family. The encoding method provided by the present invention is particularly suitable for genotyping specific target genes. At this time, at least one pair (often a pair or a few pairs) of primers can be used for amplification, and at least 2 probes can be used to detect the difference between the primers. The sequence achieves the purpose of genotyping. the

Said probes are preferably probes independent of polymerase 5'→3' exonucleic acid activity, such as molecular beacon probes and displacement probes. the

Said real-time nucleic acid amplification detection is the genotyping of real-time nucleic acid amplification detection, and the real-time nucleic acid amplification is selected from one of real-time PCR, real-time RT-PCR, Q-Beta amplification, and real-time nucleic acid sequence-dependent amplification . the

When single-color and multi-color probes co-encode multiple alleles, the distinction between homozygous and heterozygous is by adjusting the proportion of probes labeled with each fluorescent basic element in the multi-color probe to make the multi-color probe detectable When homozygous, the peak values of the fluorescence curves of each color are basically the same. In this way, when the two alleles detected by the single-color and multi-color probes coexist in a heterozygote, the fluorescence peak of that color in the single-color probe will be higher than that of other colors. the

Said probes are preferably highly specific, target-sequence-specific fluorescent probes that can distinguish only a single base difference. This kind of probe is suitable for the identification of differences in various target sequences, and the coexistence of a large number of probes will not cause interference between each other. Only the combination of the target sequence that completely matches the probe can cause the increase of the fluorescence intensity of the probe. the

In a word, the gist of the present invention is to synergistically utilize the three variables of the number of fluorescent basic elements, the cycle threshold ( _CT value) and the relative intensity of each fluorescent basic element to generate any number of gene (signal) specific fluorescent fingerprints. In order to detect many different nucleic acid sequences at the same time under the condition that the number of fluorescent basic elements is limited.

It can be seen that, according to the probe coding method, no matter using combination coding, permutation coding or index coding, the number of codes far more than that of fluorescent basic elements can be obtained, so that more types of target sequences can be detected. the

Probes labeled with one fluorescent dye (ie, one fluorescent element) may be referred to as monochromatic fluorescent probes or monochromatic probes. Similarly, two or more fluorescent dyes (that is, multiple fluorescent basic elements) can be compounded or mixed with labeled probes, which are called composite multicolor fluorescent probes or multicolor probes (see Figure 1). Both single-color probes and multi-color probes are designed for their respective target sequences, that is to say, they can detect their respective target sequences. The sequences of single-color probes or multi-color probes can be the same or different, but in most cases they are the same. the

The present invention is suitable for genotyping based on real-time nucleic acid amplification detection, real-time nucleic acid amplification includes real-time PCR, real-time RT-PCR, real-time nucleic acid sequence-dependent amplification (NASBA) and the like. Of course, the present invention does not exclude the detection of terminal nucleic acid amplification. However, the inventors believe that real-time detection can give full play to the advantages of the present invention. First of all, the real-time detection method is a kinetic detection method, even if a large number of probes coexist and cause an increase in the background signal, it will not have a significant impact on the kinetic detection characteristics. Secondly, the cycle threshold (C _T ) of the reaction curve of real-time nucleic acid amplification detection such as real-time PCR detection is determined by the template concentration and has excellent reproducibility. For multicolor probes designed with the same sequence, each fluorescent The amplification curves displayed by the dyes should have the same or at least similar C _T values. In addition, for array-coded and index-coded multicolor probes, the judgment results are based on the relative position and intensity of peaks among multiple real-time amplification curves displayed by the multicolor probe in the same reaction (fluorescence fingerprint ). Using multicolor probes of the same sequence, although the fluorescent dyes are different, the efficiency of hybridization with the template should be the same, and the degree of external influence should be the same. Therefore, the final fluorescence of the real-time amplification curve of the multicolor probes of the same sequence The ratio of intensities will also be relatively constant. This allows us to use the specific fluorescent fingerprints generated by the hybridization of each combination of multicolor probes with its specific sequence to distinguish which specific sequence exists in the PCR product and can be amplified.

Although the probe coding method described in the present invention is focused on being applied to nucleic acid detection, any person in the industry who understands basic common sense and technology can extend it to other fluorescent detection systems and even non-fluorescent coding systems according to the idea of the present invention, so as to achieve limited utilization. Several elements to encode an infinite number of signature markers. Therefore, the invention covers but should not be limited to the field of nucleic acid detection. the

It can be seen that, compared with the current general method of using a fluorescent basic element to label a target sequence-specific probe, the present invention can label more specific indicator probes by combining with each other, thereby detecting more target sequence. the

Description of drawings

Figure 1 is a schematic diagram of the multicolor probe. In Figure 1, FAM (open circle) and ROX (solid circle) are two fluorescent dyes, coded 1000 and 0100, respectively. After they are mixed with each other, it can be considered that a new type of dye is formed, that is, a mixed dye. As shown in FAM/ROX in Figure 1 (hollow and solid half circles), the code is 1100. Similarly, if the same probe is labeled with two dyes, as shown in Figure 1, the probes are labeled with FAM (open circle) and ROX (solid circle), after the two are mixed, it is equivalent to forming a A probe labeled with a mixture of dyes, here a dual-color probe. The probe is labeled with FAM/ROX ((open and half half circles, code 1100). The gray circles indicate the quenchers that replace the probes. 

Fig. 2 is the real-time PCR typing of 8 strains (strains 1-8) standard strains of Bacillus cereus. In Fig. 2, fluorescence detection is carried out simultaneously in four channels of Cy5 (solid sphere), ROX (hollow sphere), HEX (solid triangle) and FAM (open triangle). The abscissa is Cycle Number, and the ordinate is Fluorescence.

Fig. 3 is the typing result of HPV15 genotypes. In Fig. 3, fluorescence detection is carried out simultaneously in four channels of Cy5 (solid sphere), ROX (hollow sphere), HEX (solid triangle) and FAM (open triangle). The abscissa is Cycle Number, and the ordinate is Fluorescence. the

Fig. 4 is the real-time RT-PCR typing system of 6 genotypes of HCV. In Fig. 4, fluorescence detection is carried out simultaneously in four channels of Cy5 (solid sphere), ROX (hollow sphere), HEX (solid triangle) and FAM (open triangle). The abscissa is Cycle Number, and the ordinate is Fluorescence. the

Detailed ways

The following embodiments will further illustrate the present invention in conjunction with the accompanying drawings. In fact, the following embodiments just illustrate the present invention, but are obviously not limited to these applications. People in the industry can fully apply the principles of the present invention to multiple fields. the

Example 1: Probe encoding method for genotyping of Bacillus cereus. the

Bacillus cereus is a foodborne pathogen that can cause infectious diarrhea and is also the most common opportunistic pathogen that causes bacterial food poisoning. How to determine the source of infection in time is the key to control and reduce the occurrence of such bacterial food poisoning. At present, the traceability of Bacillus cereus food poisoning is still based on traditional biochemical typing, but biochemical typing takes a long time, the results are unstable, lack of reproducibility, and all strains cannot be typed, so it cannot be timely Traceability. the

The vrrA gene used for typing in this embodiment is a polymorphic site of Bacillus anthracis, which has a tandem repeat sequence and a hypervariable region. Since Bacillus cereus and Bacillus anthracis belong to Bacillus, the vrrA gene is used as Molecular genetic markers were used to study the polymorphisms of Bacillus cereus, and real-time PCR technology and combined probe coding technology were used for genotyping. the

First, fluorescent double-strand replacement probes were designed according to the vrrA gene sequences of 8 standard strains. Each strain had a set of specific probes, which were labeled with different combinations of four fluorescent dyes, FAM, HEX, ROX, and CY5. Code it (see Table 1). Then PCR amplification was performed with a pair of primers reported in the literature, and the product was 230 bp. The primer sequences were: Sense: 5'-ACTACCACCAATGGCACA-3', Anti-sense: 5'-GCTGCATGTATGGTTGAT-3'. the

In the same PCR reaction tube, add all probes, the total reaction volume is 25 μL, containing 2.5 μL of 10X buffer solution, 3.0 mM MgCl ₂ , 0.25 mM dNTP, 1.0 U Taq enzyme, 20 pmol upstream primer, 2.5 pmol downstream primer, probe Needle 20pmol each, 5μL bacterial liquid template. Reaction conditions: 95°C for 3min, the first cycle is 94°C for 15s, 52°C for 20s, 72°C for 20s, a total of 10 cycles, the second cycle is 94°C for 15s, 52°C for 20s, 72°C for 20s, 28°C 18s, a total of 40 cycles, the fluorescence is collected at 28°C in the second cycle. MX3000P real-time PCR instrument was used for detection. In order to improve the fluorescence efficiency and hybridization efficiency of the probes, low-melting point probes and asymmetric PCR were used in the experiment; finally, according to the real-time fluorescent PCR results (differences in fluorescence curves), 8 standard strains could be successfully divided into 8 subgroups. type (see Figure 2).

Example 2: Genotyping of Human Papillomavirus (HPV). the

HPV is a group of highly specific DNA viruses. The viral nucleic acid is double-stranded circular DNA, containing 8000 base pairs, belonging to subgroup A of Papovaviridae, with a diameter of about 55nm. HPV has a strong tropism for the squamous epithelium of the reproductive tract. The gene structure of various HPVs is roughly similar and can be divided into three parts: late zone, early zone and regulatory zone. HPV is related to the occurrence of many diseases, and serious cases can cause cancer, such as cervical cancer and skin cancer. According to the relationship between HPV and tumorigenesis, it can be divided into high-risk and low-risk types. In this embodiment, 15 different types of HPV are used as experimental objects, and fluorescent double-strand displacement probes are designed according to the gene sequence of the L1 region, and a set of specific probes (Table 2) are designed for each type, labeled FAM, All combinations of the four fluorescent dyes HEX, ROX, and CY5, and are coded in binary. A pair of universal primers GP5+/GP6+ was used for PCR amplification, and the product was about 150 bp. The primer sequence was GP5+: 5′-TTTGTTACTGTGGTAGATACTAC-3′, GP6+: 5′-GAAAAATAAACTGTAAATCATA-TTC-3′. In a PCR tube, add all probes, the total reaction volume is 25 μL, containing 50 mM KCl, 2.5 mM MgCl ₂ , 0.25 mM dNTP, 1.0 UTaq enzyme, 0.2 μM of upstream and downstream primers, 0.2 μM of each probe, 5 μL plasmid template. The reaction conditions were: 95°C for 3 minutes, the cycle period was 94°C for 15s, 45°C for 20s, and 72°C for 20s, a total of 40 cycles, and the fluorescence was collected during annealing. Detection was carried out with Rotor-Gene3000 real-time PCR instrument. Finally, according to the real-time fluorescent PCR results (differences in fluorescence curves), 15 different subtypes of HPV viruses were successfully distinguished (see FIG. 3 ).

Embodiment 3: HCV has a high degree of variability, and various types are widely distributed all over the world. At the same time, the popular genotypes in specific areas are also found in other areas, which makes the distribution of HCV genotypes complicated. In addition, the continuous improvement of HCV genotyping means Developed and refined, so that reports of genotype distributions also appear frequently. In the example, six main subtypes in China: 1b, 2a, 3a, 1a, 3b and 6a were taken as objects, and they were classified according to the principle of probe coding. For the 6 genotypes of HCV, only 3 fluorescent dyes need to be selected to form six combinations of FAM, HEX, ROX, FAM+HEX, HEX+ROX, and FAM+ROX. The probe sequences are shown in Table 3. The 6 common genotypes of HCV in China are coded and distinguished. the

Utilize universal primers to carry out RT-PCR amplification, upstream primer 5'-CCCICGITTGGGTGTGCG-3', downstream primer: 5'-GTTAGGGTATCGATGACITTAC-3', wherein I is inosine nucleotide, and the length of the universal primer PCR amplification product is 256_bp . Perform RT-PCR genotyping in the same reaction tube with universal primers, the total reaction volume is 30μL, containing 50mM KCl, 2.5mM MgCl2, 0.25mM dNTP, 1.0UTaq enzyme, 12U M-MLV reverse transcriptase, 20URNAsin, 0.2 μM upstream primers, 0.2 μM downstream primers, 0.2 μM each probe, add 5uL and thermally lyse pseudoviruses of each genotype at 95°C for 5 minutes as RNA templates. Reaction conditions: 42°C, 40 min, 95°C for 3 min, 40 cycles in total at 95°C for 15 s, 50°C for 20 s, and 72°C for 20 s, and the fluorescence signal was collected at 50°C. Finally, according to the real-light PCR results, six different HCV subtypes can be successfully distinguished (see FIG. 4 ). the

Example 4: Probe combination coding method for multiplex real-time nucleic acid amplification detection. If there are A element and B element cable, three combinations of A, B, and A+B can be formed respectively; similarly, if there are three elements A, B, and C, A, B, C, A + B, and A + C can be formed , B+C, A+B+C a total of seven combinations. Therefore, according to the mathematical combination rules, for N fluorescent basic elements, there are altogether C _N ¹ +C _N ² +…+C _N ^N =∑C _N ⁱ (i=1～N)=2 ^N -1 combinations , any of which can be labeled with a target sequence-specific probe, so 2 ^N -1 fluorescent probes with different labels can be obtained. This allows detection of ^2N -1 different target sequences. According to this scheme, the four-color fluorescent PCR instrument (which can simultaneously detect four fluorescent basic elements) can detect 2 ⁴ -1 = 15 kinds of target sequences, and the five-color fluorescent PCR instrument (which can simultaneously detect five fluorescent basic elements) 2 ⁵ -1=31 genotypes can be detected.

According to the combination coding method, it can be seen that the current measurement method actually only uses a single fluorescent basic element to label the probes one by one, without considering the combination between them, and the encoded probes can only be equal to the number of fluorescent basic elements . Therefore, for a four-color fluorescent PCR instrument (which can detect four fluorescent basic elements in total), it can only encode four fluorescent probes and detect four target sequences. Similarly, a five-color fluorescent PCR instrument (which can detect five fluorescent basic elements in total) can only detect five target sequences at the same time. the

Example 5: Probe array encoding method for multiplex real-time nucleic acid amplification detection. The combination coding method only considers the different combinations of fluorescent basic elements and does not consider whether the relative intensities of the fluorescent basic elements are the same. Composition of different arrangements, for example, the same combination code composed of A+B, can be distinguished as A>B or A<B according to the difference in fluorescence intensity between them (although A=B represents the third case , but because it is very difficult to control the intensity of two or more fluorescent basic elements in the PCR reaction to be the same, and when A=B, AB and BA cannot be distinguished, and cannot be expressed in an arrangement, so this situation is not included in the arrangement code Prediction and consideration within the scope), using these two situations to represent two kinds of probes respectively, two kinds of codes can be obtained. Also combined by A+B+C combination code, A>B>C, A>C>B, B>A>C, B>C>A, C>A>B, C can be formed according to the difference in fluorescence intensity >B>A and other 6 cases, can get 6 codes. This combination conforms to the arrangement rules of mathematics. Therefore, for N fluorescent basic elements, ΣP _N ⁱ (i=1˜N) different combinations of fluorescent basic elements can be obtained. Any of them can be labeled with a target sequence-specific probe, so that ΣP _N ⁱ (i=1～N) different target sequences can be detected. According to the sequence code, the four-color fluorescent PCR instrument can simultaneously detect P ₄ ¹ +P ₄ ² +P ₄ ³ +P ₄ ⁴ = 64 target sequences, and the five-color fluorescent PCR instrument can simultaneously detect P ₅ ¹ +P ₅ ² +P ₅ ³ + P ₅ ⁴ + P ₅ ⁵ = 325 target sequences.

Example 6: Probe Index Encoding Method for Multiplex Real-Time Nucleic Acid Amplification Detection. It is not difficult to see that the permutation coding method only considers the size difference between the fluorescent basic elements, and does not consider the degree of the size difference. If we divide the size of each fluorescent basic element into several levels, which are called fluorescence levels, and use x to represent the number of fluorescence levels, assuming that the x values of different fluorescent groups are equal, then for N-color fluorescent PCR instruments, we can design The number of probes is: P _N ⁱ +∑ _X ⁱ P _N ⁱ (i=2～N) fluorescent probes (this formula counts the level number of monochromatic fluorescent probes as 1, which is considering that the fluorescence intensity itself is relative to this property). According to this rule, if the number of fluorescent levels of a dye x=10, then the four-color fluorescent PCR instrument can simultaneously detect P ₄ ¹ +(10 ² P ₄ ² +10 ³ P ₄ ³ +10 ⁴ P ₄ ⁴ )=265 , 204 genotypes, since theoretically the number of fluorescence levels of each fluorescent dye can be arbitrarily many, so any number of coding schemes can be designed theoretically for index coding.

Embodiment 7: The said fluorescent probe of the present invention refers to the fluorescent probe that can be used for real-time nucleic acid amplification detection, and they include TaqMan ^TM probe, TaqMan-MGB ^TM probe, molecular beacon probe (Molecular Beacons) , Fluorescence energy resonance transfer probes (also known as LightCycler ^TM probes), displacement probes, Scorpions primers (Scorpions), Amplifier primers, etc. These fluorescent probes can be divided into probes that depend on the 5′→3′ exonucleic acid activity of the polymerase, such as TaqMan probes, and probes that do not depend on the 5′→3′ exonucleic acid activity of the polymerase, such as molecular beacons. probes and displacement probes, etc.

Example 8: The present invention preferably uses probes independent of polymerase 5'→3' exonucleic acid activity, such as molecular beacon probes and displacement probes. Because these probes can be used in all modes of real-time nucleic acid amplification detection, and the reaction conditions are not limited by the 5'→3' exonuclease activity of nucleic acid polymerase. the

Example 9: The present invention preferably uses highly specific fluorescent probes, which can distinguish target sequences with only a single base difference. It is suitable for the identification of various target sequence differences, and the coexistence of a large number of probes will not cause mutual interference. Because fluorescent probes that differ by a single nucleotide can be distinguished, only binding to a target sequence that exactly matches the probe can cause an increase in the fluorescence intensity of the probe. the

Example 10: The encoding method provided by the present invention is suitable for the detection of multiple target sequences, especially suitable for detecting genotypes that may only contain one (or a few) of the multiple target sequences in the sample. Especially for the genotyping of different alleles in the same gene (or gene family), since one or a few pairs of primers can be used to amplify, and the encoded fluorescent probes can be used to identify the sequences between different primers, the Mix all probes in the same tube. Using one or a few pairs of primers has less chance of non-specific amplification, and the reaction conditions are easier to optimize. For the reaction system with a large number of probes, it means that the optimization of reaction conditions can be concentrated on the probes, so that The establishment process of the detection system is simpler. the

Example 11: The present invention is particularly suitable for genotyping a specific target gene. At this time, one or a few pairs of primers can be used for amplification, and multiple probes can be used to detect the intermediate sequence of the primers to achieve the purpose of genotyping. For example, real-time PCR typing of human papillomavirus (HPV), real-time RT-PCR typing of immunodeficiency virus (HIV), real-time RT-PCR typing of hepatitis C virus, and 16S ribosomal RNA of bacteria real-time PCR typing, or real-time PCR typing for a certain type of bacteria, including drug-resistant mutation typing, etc. the

Example 12: When the wooden invention is used for genotyping a specific target gene, in the case where two or more subtypes may coexist in the same specimen, the real-time PCR curve of the two probes can be used to C _T value, relative fluorescence intensity and the shape of the amplification curve are distinguished. For FAM and HEX-encoded dual-color probes (encoding the same target sequence), when the corresponding target sequence of the dual-color probe exists in the reaction, the C _T of the two real-time PCR curves displayed on the FAM and HEX channels on the instrument The difference (ΔC _T ) should be zero theoretically, and the ratio of the fluorescence intensities of the two is constant. If two different target sequences encoded by the FAM monochrome probe and the HEX monochrome probe appear at the same time, the ΔC _T of the real-time amplification curves of the two will be either positive or negative, and the ratio of the fluorescence intensities of the two It must also be different from the case of two-color probes. Only when the starting target sequence content of the target sequence detected by the dual-color probe and the target sequence detected by the two single-color probes is exactly equal (such as two different alleles in heterozygotes, ΔC _T = 0 appears at this time) , and when the fluorescence intensity ratio of the two detected by the instrument is exactly equal to the fluorescence ratio of the two-color probe, the two indistinguishable results will be caused. Obviously, the probability of the latter two cases occurring at the same time is extremely low. Because the possibility of the subtype content in the mixed sample is completely equal is very small, the fluorescence intensity of the fluorescent probe is affected by many factors, and the ratio of the fluorescence intensity of the two single-color probes is exactly equal to the possibility of the fluorescence ratio of the two-color probe less sexual. The normal real-time PCR amplification curve belongs to a typical S-type, and no sudden increase (that is, an inflection point) will occur. However, if the target sequences recognized by the single-color probe and the dual-color probe coexist, for example, the target sequences recognized by FAM and FAM/HEX coexist, the amplification curve of FAM actually reflects the sum of the two amplification curves , for example, the first half of the curve is the case of the single-color probe FAM, while the second half has both the growth of the fluorescence level of the FAM single-color probe and the growth of FAM in the FAM/HEX dual-color probe, thus appearing in the curve Growth inflection point; an inflection point occurs in the FAM amplification curve regardless of the concentration of the two target sequences. It’s just that when the concentration of the target sequence detected by the monochrome probe is high, the FAM amplification curve appears first and the HEX amplification curve appears later, and the inflection point of the FAM amplification curve must appear near the beginning of the HEX curve, because the position of the inflection point It is also the position where HEX is added. Conversely, if the concentration of the target sequence detected by the dual-color probe is high, the FAM and HEX amplification curves will first appear at the same time, but the FAM curve will then appear a growth inflection point, while the HEX curve is a typical S-shaped curve without an inflection point.

Therefore, for a system with a two-color amplification curve, it is easy to judge based on the relationship and shape (fluorescence fingerprint) between the curves. Taking FAM and HEX amplification curves in the reaction at the same time as an example, there may be five situations for the target sequence in the system: only the target sequence recognized by the two-color probe FAM/HEX exists, and there are both FAM monochrome probe and HEX monochrome probe. There are two target sequences recognized by the needle, there are two target sequences recognized by the FAM single-color probe and the two-color probe FAM/HEX, and there are two target sequences recognized by the HEX single-color probe and the two-color probe FAM/HEX, There are three kinds of target sequences recognized by FAM single-color probe, HEX single-color probe and double-color probe FAM/HEX. If the two curves belong to a typical S-type amplification curve, and ΔC _T = 0, and the fluorescence intensity ratio of the two is consistent with the amplification curve of the two-color probe, it can be directly judged that only the two-color probe FAM/HEX recognition exists the target sequence. If the two amplification curves are separated from each other and are typical S-shaped amplification curves, it can be directly judged that there are two target sequences respectively recognized by FAM and HEX monochromatic probes. If there is a new growth inflection point in the amplification curve of FAM in the two amplification curves, it means that there are two target sequences recognized by the FAM single-color probe and the two-color probe FAM/HEX at the same time. If there is a new growth inflection point in the amplification curve of HEX in the two amplification curves, it can be judged that there are two target sequences recognized by the HEX single-color probe and the two-color probe FAM/HEX at the same time. If there are new growth inflection points in the amplification curves of FAM and HEX in the two amplification curves, it can be judged that there are three types of FAM single-color probes, HEX single-color probes, and two-color probes FAM/HEX recognition. target sequence.

Of course, for high-volume genotyping situations, the probability of interference increases. However, as long as we make full use of the three variables, the number of fluorescent basic elements, the _CT value, and the relative intensity of each fluorescent basic element, many genotype-specific fluorescent fingerprints can be generated. Then by fully grasping the clinical data and appropriately changing the probe labeling strategy, it is entirely possible to rule out the interference caused by coexisting subtypes.

Embodiment 13: When the present invention is used to detect one (homozygous) or two (heterozygous) situations in a plurality of alleles that may exist simultaneously in a sample, if an allele uses a single color ( For example, the red) probe is used for detection and the other allele is detected with a dual-color or multi-color probe containing the color of the above-mentioned single-color probe. At this time, since ΔC _T =0, it is necessary to distinguish heterozygotes from homozygotes Depending on the change in fluorescence intensity. In order to facilitate the distinction, it is necessary to adjust the proportion of probes labeled with the fluorescent basic elements of each color in the two-color or multi-color probe, so that the peak values of the fluorescence curves of each color are basically equal when the two-color or multi-color probe detects homozygous samples; thus, When different alleles that can be detected by single-color (such as red) probes and bicolor (such as red, green) or multicolor (such as red, green, yellow) probes coexist in a heterozygote, the red probe Although the peaks are produced simultaneously with probes of other colors, their peaks must be higher than those of other colors, so that the homozygotes detected by single-color probes (only one-color fluorescence appears) and those detected by two-color or multi-color probes Homozygotes (there are two-color or multi-color curves peaking at the same time and the peaks of each curve are basically the same) and single-color—heterozygotes detected by two-color (multi-color) probes (two-color or multi-color curves are peaking at the same time but The peak of a certain color curve will be significantly higher than the peak of other curves). Using the same principle, homozygotes and heterozygotes detected by different multicolor probes can also be distinguished.

Nucleotide sequence list

Table 1: Probe sequences and codes in vrrA genotyping

Table 2 HPV subtype-specific probes and their codes

Table 3: HCV subtype-specific probes and their codes

Note: The characters with borders are bases that do not match the template, which can reduce the quenching effect of bases on fluorescein HEX during hybridization.

Claims (9)

1. one kind is used for the probe coding method that multiplex real-time nucleic acid amplification detects, and it is characterized in that the steps include:

The different fluorescence dyes that 1) the nucleic acid amplification detecting instrument can be detected are referred to as the fluorescence fundamental element as the fundamental element of fluorescent probe mark;

2) coming the special indication probe of compound or mixed mark target sequence with the mutual combination between the fluorescence fundamental element, is not the special indication probe that single fluorescence fundamental element comes compound or mixed mark target sequence with the mutual combination between the fluorescence fundamental element; The probe of institute's mark is joined the real-time nucleic acid amplification system and determined nucleic acid sequence is increased; Gene specific fluorescence finger printing and corresponding target sequence thereof are distinguished, identified to collaborative kind, cycle threshold difference and each these three variable of fluorescence fundamental element relative intensity difference of the fluorescence fundamental element that occurs in the real-time nucleic acid amplification curve spectrum of utilizing, so that in the situation that the limited a kind of sequence that detects in the multiple different IPs acid sequence of the basic element number of fluorescence;

Said fluorescence fundamental element is ALEXA 350, FAM, HEX, TET, JOE, VIC, ROX, Texas Red, Cy5, Cy5.5 or TAMRA.

2. the probe coding method that detects for multiplex real-time nucleic acid amplification as claimed in claim 1, it is characterized in that the said assembly coding that mutually is combined as, assembly coding only considers the difference of the basic Element Species class of fluorescence and ignores the difference of fluorescence intensity between different fluorescence fundamental elements, and the fluorescence fundamental element is combined into different fluorescent mark group or probe by the combining rule of mathematics.

3. the probe coding method that detects for multiplex real-time nucleic acid amplification as claimed in claim 1, it is characterized in that the said arranging and encoding that mutually is combined as, arranging and encoding not only considered the difference of the basic Element Species class of fluorescence but also distinguished relatively strong and weak between each fluorescence fundamental element, was combined into different fluorescent mark group or probe by the queueing discipline of mathematics.

4. the probe coding method that detects for multiplex real-time nucleic acid amplification as claimed in claim 1, it is characterized in that the said index coding that mutually is combined as, index coding is considered the relatively power of fluorescence intensity between the difference, each element of the basic Element Species class of fluorescence and fluorescence intensity difference in size in various degree simultaneously, is combined into different fluorescent mark group or probe by the rule of exponentials on the mathematics.

5. the probe coding method for the multiplex real-time nucleic acid amplification detection as claimed in claim 1 is characterized in that the fluorescent probe that said fluorescent probe detects for being used for real-time nucleic acid amplification, is selected from TaqMan ^TMProbe, TaqMan-MGB ^TMProbe, molecular beacon probe, fluorescent energy resonance transfer probe, displacement probe, scorpion primer or Amplifier primer.

6. the probe coding method for the multiplex real-time nucleic acid amplification detection as claimed in claim 1 it is characterized in that it is that the polychrome real-time nucleic acid amplification detects that said nucleic acid amplification detects, or the nucleic acid amplification of terminal point formula detects.

7. the probe coding method that detects for multiplex real-time nucleic acid amplification as claimed in claim 6, it is characterized in that said real-time nucleic acid amplification detects the gene type that detects for real-time nucleic acid amplification, real-time nucleic acid amplification is selected from a kind of in the amplification of PCR in real time, real-time RT-PCR, Q-Beta amplification, the dependence of real-time nucleic acid sequence.

8. the probe coding method for the multiplex real-time nucleic acid amplification detection as claimed in claim 1 is characterized in that the kind of said fluorescence fundamental element refers to have those fluorescence fundamental elements to occur in the real-time nucleic acid amplification curve spectrum; The difference of appearance time between each fluorescence fundamental element that said cycle threshold difference refers to occur; Fluorescence intensity difference in size degree refers to the big or small level of each fluorescence fundamental element between the said fluorescence fundamental element, or fluorescence level, or relative extent, or relative intensity is combined into different elements.

9. the probe coding method that detects for multiplex real-time nucleic acid amplification as claimed in claim 1 is characterized in that utilizing various fluorescence fundamental elements to go out the relative time at peak, the sort of sequence existed and augmentation detection in addition when curve shape was distinguished multiple target sequence coexistence.

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